Immunochemical Difference of Wound-Induced 1-Aminocyclopropane-1-carboxylate Synthase from the Auxin-Induced Enzyme

Immunochemical cross-reactivity of wound- and auxin-induced1-aminocyclopropane-1-carboxylate (ACC) synthase was examinedwith the antibody against wound-induced ACC synthase purifiedfrom mesocarp of winter squash (Cucurbita maxima Duch.). Theantibody recognized ACC synthase from wounded hypocotyls ofwinter squash and from wounded pericarp of tomato fruits, butnot the enzyme from IAA-treated hypocotyls of winter squash,tomato and mung bean. These results indicate that the primarystructure of the wound-induced enzyme is different from thatof the auxin-induced enzyme in the same species, and impliesthat there are two different genes for ACC synthase, one forwound induction and the other for auxin induction. (Received June 14, 1988; Accepted July 20, 1988)     

Particulate cytochromes of mung bean seedlings

Efforts have been made to solubilize cytochrome components from particulate fractions of etiolated mung bean seedlings. Low temperature spectrophotometry reveals that the cytochrome composition of mitochondria isolated from whole seedlings is the same as that reported by Bonner for mung bean hypocotyls. On the basis of the identity in position of the α-bands in low temperature difference spectra for mitochondria, for a partially purified haemoprotein from mitochondria, and for purified cytochrome b-555, it is suggested that cytochrome b-555 is an intrinsic component of mung bean mitochondria. Difference spectra show that both the mitochondrial and microsomal fractions contain at least 2 b-type cytochromes. Cytochrome b-555 is almost certainly present in the microsomes, since the low temperature difference spectrum for the cytochrome is identical with the spectrum for this particulate fraction.

By freezing and thawing mung bean mitochondria in 4% cholate and centrifuging, cytochrome oxidase activity can be concentrated in the supernatant fraction, although it is not completely solubilized. The oxidase is inhibited by high concentrations of cytochrome c. A particle-bound cytochrome c can be obtained from mitochondria by digestion with snake venom. However, the autoxidizability of the preparation indicates that the cytochrome has been solubilized in a modified form. A CO-binding pigment can be obtained from mung bean microsomes by digestion with snake venom.

     

A Vacuolar Processing Enzyme in Maturing and Germinating Seeds: Its Distribution and Associated Changes during Development

Proprotein precursors of vacuolar components are transportedfrom endoplasmic reticulum to the dense vesicles, and then targetedto the vacuoles, where they are processed proteolytically totheir mature forms by a vacuolar processing enzyme. Immunoelectronmicroscopy of the maturing endosperm of castor bean (Ricinnscommunis) revealed that the vacuolar processing enzyme is selectivelylocalized in the dense vesicles as well as in the vacuolar matrix.This indicates that the vacuolar processing enzyme is transportedto vacuoles via dense vesicles as does IIS globulin, a majorseed protein. During seed maturation of castor bean, an increasein the activity of the vacuolar processing enzyme in the endospermpreceded increases in amounts of total protein. The enzymaticactivity reached a maximum at the late stage of seed maturationand then decreased during seed germination concomitantly withthe degradation of seed storage proteins. We examined the distributionof the enzyme in different tissues of various plants. The processingenzyme was found in cotyledons of castor bean, pumpkin and soybean,as well as in endosperm, and low-level processing activity wasalso detected in roots, hypocotyls and leaves of castor bean,pumpkin, soybean, mung bean and spinach. These results suggestthat the proprotein-processing machinery is widely distributedin vacuoles of various plant tissues. (Received July 11, 1993; Accepted August 17, 1993)     

Inhibition of auxin-induced ethylene production by salicylic acid in mung bean hypocotyls

Salicylic acid (SA), a common plant phenolic compound, influences diverse physiological and biochemical processes in plants. To gain insight into the mode of interaction between auxin, ethylene, and SA, the effect of SA on auxininduced ethylene production in mung bean hypocotyls was investigated. Auxin markedly induced ethylene production, while SA inhibited the auxin-induced ethylene synthesis in a dose-dependent manner. At 1 mM of SA, auxininduced ethylene production decreased more than 60% in hypocotyls. Results showed that the accumulation of ACC was not affected by SA during the entire period of auxin treatment, indicating that the inhibition of auxin-induced ethylene production by SA was not due to the decrease in ACC synthase activity, the rate-limiting step for ethylene biosynthesis. By contrast, SA effectively reduced not only the basal level of ACC oxidase activity but also the wound-and ethylene-induced ACC oxidase activity, the last step of ethylene production, in a dose-dependent manner. Northern and immuno blot analyses indicate that SA does not exert any inhibitory effect on the ACC oxidase gene expression, whereas it effectively inhibits both the in vivo and in vitro ACC oxidase enzyme activity, thereby abolishing auxin-induced ethylene production in mung bean hypocotyl tissue. It appears that SA inhibits ACC oxidase enzyme activity through the reversible interaction with Fe²⁺, an essential cofactor of this enzyme. These results are consistent with the notion that ethylene production is controlled by an intimate regulatory interaction between auxin and SA in mung bean hypocotyl tissue.     

Isolation of Smooth Endoplasmic Reticulum and Tonoplast from Mung Bean Hypocotyls (Vigna radiata [L.] Wilczek) Using a Ficoli Gradient and Two-Polymer Phase Partition

A new method for the isolation of smooth endoplasmic reticulumand tonoplast from etiolated mung bean hypocotyls (Vigna radiata[L.] Wilczek) has been developed. After centrifugation in aFicoli density gradient [5.5% (w/w) in 15% (w/w) sucrose] ofa crude microsomal membrane fraction (10,000–156,000?gpellet) which had been prepared and resuspended in buffer systemsthat contained 0.25 M sorbitol, more than 80% of the total amountsof smooth endoplasmic reticulum and tonoplast were co-bandedat the interface between the sample load and the Ficoll layer,while most of the other cellular membranes, including plasmamembrane, Golgi membranes and yellow-colored membrane materials,which were presumably the etioplast envelopes, were sedimentedthrough the Ficoli layer. Smooth endoplasmic reticulum and tonoplastwere separated from each other to a high degree of enrichmentby a subsequent two-polymer phase partitioning. The separationis based on the principle that mung bean tonoplast has a highpartition coefficient for the polyethylene glycol-enriched upperphase and the smooth endoplasmic reticulum has a high partitioncoefficient for the Dextran-enriched lower phase. Assessed interms of degree of contamination by activities of membrane markerenzymes, the isolated smooth endoplasmic reticulum and tonoplastfractions were found to be highly purified. An ATPase sensitiveto neutral detergent and vanadate was found to be specificallyassociated with the smooth endoplasmic reticulum. ¹Contribution No. 3172 from the Institute of Low TemperatureScience (Received April 22, 1988; Accepted September 28, 1988)     

Changes in mitochondrial activity during enlargement of mung bean root cells

A mitochondrial fraction was separately prepared from two differentregions of mung bean roots; from root-tips which contained mainlyimmature cells and from tissue, other than the root-tips, whichwas composed of mature and fully vacuolated cells. The malatedehydrogenase, fumarase and aconitase activities per cell didnot increase or did so only slightly during cell growth. Respiratoryactivity of both tissue sections and the crude mitochondrialfraction also seemed to increase slightly as cells matured.However, the cytochrome oxidase and succinate cytochrome c reductaseactivities per cell increased significantly during cell enlargement.There was no difference in the distribution-profiles of thecytochrome oxidase and malate dehydrogenase activities aftersucrose density gradient centrifugation, between mitochondrialfractions prepared from two regions of the. roots. The malatedehydrogenase activity per unit of cytochrome oxidase activityin purified mitochondria of immature cells was much higher thanthat of mature cells. The results suggest that enzymes in mitochondrialmatrix are mainly synthesized in immature cells or during celldivision. In contrast, enzymes in the cristae seem to be formedduring cell maturation, as well as being formed in immaturecells. (Received January 26, 1973; )     

Subunit composition of vacuolar membrane H(+)-ATPase from mung bean

The vacuolar H(+)-ATPase from mung bean hypocotyls was solubilized from the membrane with lysophosphatidycholine and purified by QAE-Toyopearl column chromatography. The purified ATPase was active only in the presence of exogenous phospholipid and was inhibited by nitrate, dicyclohexyl carbodiimide and Triton X-100, but not by vanadate or azide. Dodecyl sulfate/polyacrylamide gel electrophoresis of the purified ATPase yielded ten polypeptides of molecular masses of 68 kDa, 57 kDa, 44 kDa, 43 kDa, 38 kDa, 37 kDa 32 kDa, 16 kDa, 13 kDa and 12 kDa. All polypeptides remained in the peak activity fraction after glycerol density gradient centrifugation. Nine of them, excluding the 43-kDa polypeptide, comigrated in a polyacrylamide gradient gel in the presence of 0.1% Triton X-100. The 16-kDa polypeptide could be labeled with [14C]dicyclohexylcarbodiimide. The amino-terminal amino acid sequence of the isolated 68-kDa polypeptide generally agreed with that deduced from the cDNA for the carrot 69-kDa subunit [Zimniak, L., Dittrich, P., Gogarten, J. P., Kibak, H. & Taiz, L. (1988) J. Biol. Chem. 263, 9102-9112]. Thus, mung bean vacuolar H(+)-ATPase seems to consist of nine distinct subunits.     

Ferulic acid impairs rhizogenesis and root growth,and alters associated biochemical changes in mung bean (Vigna radiata) hypocotyls

The present study investigated the effect of ferulic acid (FA; 0–1000 µM) on early growth, and rhizogenesis in mung bean (Vigna radiata) hypocotyls and associated biochemical changes. FA severely affected the radicle elongation and number of secondary roots after 72 h. The root and shoot length, number and length of secondary roots, and seedling dry weight of one-week-old seedlings of mung bean were decreased by 64%. The rooting potential (percent rooting, number and length of adventitious roots) of mung bean hypocotyls under in vitro conditions was significantly inhibited in response to 1–100 µM FA. At 1000 µM there was complete cessation of rooting. FA caused a reduction in the contents of water-soluble proteins and endogenous total phenolics, whereas the activities of proteases, peroxidases, and polyphenol peroxidases increased. The study concludes that FA inhibits root growth and development, and in vitro rooting process in mung bean by interfering with biochemical processes that are crucial for root formation.     

A phosphatidyl choline exchange protein isolated from germinated castor bean endosperms

A phospholipid exchange protein (PLEP) functioning between theendoplasmic reticulum and the mitochondrion was purified fromthe cytosolic fraction of germinated castor bean endosperms.In the protein fraction eluted from Sephadex G-100 column, theexchange rate reached 7.3µg phospholipids exchanged/mgprotein/15 min, which was 60-fold that of pota to tuber PLEP.The lipid transfer by this protein was specific for phosphatidylcholine and the transfer rate from microsomes to mitochondriawas as high as that from mitochondria to microsomes. Castorbean PLEP transferred phospholipid from castor bean microsomesto mitochondria from other sources such as potato tubers, cauliflowerinflorescences, pumpkin hypocotyls and rat livers, and to liposomes,but not to Avena etioplasts. In addition, it transferred phospholipidfrom potato microsomes to potato mitochondria. (Received November 17, 1978; )     

Cyanide-insensitive Respiration in Plant Mitochondria

Pathways of electron transport have been studied in mitochondria isolated from hypocotyls of etiolated mung bean seedlings and skunk cabbage spadices that show cyanide-resistant respiratory activity. The residual flux through cytochrome c oxidase is shown to be small in comparison with the flux through an unidentified alternative oxidase that is known to have a high affinity for oxygen. This alternative oxidase is not a cytochrome. Skunk cabbage and mung bean mitochondria contain cytochromes a and a₃ that have absorption peaks differing slightly from those of animal preparations. A slow oxidation-reduction of cytochrome a₃-CN has been demonstrated. Cytochromes b undergo oxidation and reduction in the presence of cyanide but play no essential role in the cyanide-resistant pathway. Antimycin inhibits to an extent similar to that of cyanide; the respiratory chain bifurcates on the substrate side of the antimycin-sensitive site. Evidence is presented for the selective inhibition by thiocyanate, α, α′-dipyridyl, and 8-hydroxyquinoline of the alternative oxidase pathway, which may therefore contain a non-heme iron protein.     

Observations on the enzymatic synthesis of a beta-1,4-glucan by a soluble preparation form mung bean(Phaseolus aureus)

An enzyme extract of mung bean roots and hypocotyls (Phaseolus aureus) that catalyzes the synthesis of a β-1,4-glucan from guanosine 5′-diphosphate-d-glucose was prepared by a modification of the method of T.-Y. Liu and W. Z. Hassid (1970, J. Biol. Chem.245, 1922–1925). Its activity was not increased by any of those factors that have contributed to the marked improvement in the performance of various cell-free polysaccharide-synthesizing systems from other organisms. Evidence is presented to suggest that in the mung bean system a stable precursor of the cell wall polysaccharide or intermediate in its synthesis is formed by incubation of the enzyme with guanosine 5′-diphosphate-d-mannose.     

Partial Purification and Characterization of An ATPase in Mung Bean Hypocotyl Plasma Membrane: Suggestion for A New Type of Higher Plant Plasma Membrane ATPase

A vanadate-sensitive and nitrate-resistant ATPase was solubilizedwith Zwittergent 3–14 from a highly purified plasma membranefraction of mung bean hypocotyls and partially purified by glyceroldensity gradient centrifugation and phenyl-Sepharose columnchromatography. Either phosphatidylcholine or phosphatidylserinein addition to Mg^{2 +} was required for the enzyme activity, whereasK⁺, phosphatidylethanolamine and lysophosphatidylcholine hadno effect on the activity. The purified enzyme preparation containedtwo major polypeptides with molecular masses of 67 and 55 kDaas analyzed by SDS-polyacrylamide gel electrophoresis. Whenthe plasma membrane fraction was incubated with [-³²P]ATP, a45-70-kDa polypeptide(s) was labeled, and the label could berapidly chased with cold ATP. When the fraction was incubatedwith [¹⁴C]N,N'-dicyclohexylcarbodiimide, an inhibitor for theATPase, a 15-20-kDa polypeptide was labeled. We propose thatthe enzyme is a new type of higher plant plasma membrane ATP-aseand is composed of 67- and 55-kDa subunits and probably alsoa 15-20-kDa subunit. ¹Present address: Takarazuka Institute, Sumitomo Chemical IndustriesLtd., Takatsukasa, Takarazuka, Hyogo 665, Japan (Received September 2, 1987; Accepted May 20, 1988)     

Immunological Studies on ATPases in Mung Bean Hypocotyl Plasma Membrane: Proposal of the Presence of Two Molecular Species of ATPase

ATPase in a highly purified plasma membrane fraction from mungbean hypocotyls was solubilized by lysolecithin and fractionatedby glycerol density gradient centrifugation. Lysolecithin activatedATPase activity in the lower but not in the upper half of theactivity peak after glycerol density gradient centrifugation.Antibody against maize root plasma membrane ATPase [Nagao etal. (1987) Plant Cell Physiol. 28: 1181] reacted to a 100-kDapolypeptide which was localized only at the lower half of theactivity peak. Antibody against a 67-kDa polypeptide, whichwas proposed to be a subunit of a new type of ATPase in mungbean hypocotyl plasma membrane (Mito et al. the preceding paper),reacted only to its own antigen which was present mainly inthe upper half of the activity peak. The activity peak fractioncontained a low-molecular-mass polypeptide binding N.N'-dicyclohexylcarbodiimide.We propose the presence in mung bean hypocotyl plasma membraneof two distinct ATPases which differ from each other in polypeptideconstitution and in their response to lysolecithin. (Received September 2, 1987; Accepted May 20, 1988)     

Accumulation of a Glycoprotein That is Homologous to a Seed Storage Protein in Mung Bean Hypocotyls at the Late Stage of Tissue Elongation

Physiological changes were examined in the amount of a 50-kDaglycoprotein (gp50) that was recovered in a nuclear fractionfrom hypocotyls of mung bean (Vigna radiata) seedlings. Immunoblotanalysis indicated that the glycoprotein was present in hypocotylsand epicotyls from 4- and 5-day-old seedlings but not in hypocotylsfrom 2-day-old seedlings. The glycoprotein was not detectedin leaves or roots. When we divided hypocotyls of 3-day-oldseedlings into the elongating region (0 to 1.5 cm below thecotyledon) and the mature region, we found gp50 in the matureregion only. The results suggest that the 50-kDa glycoproteinis synthesized de novo and accumulates at the late stage duringelongation of cells in the hypocotyl. Furthermore, an antibodyspecific to gp50 reacted with a major 50-kDa protein in cotyledons,which is known as a storage protein in mung bean cotyledon.Eighteen amino acid residues among 22 amino-terminal residuesof gp50 were identical to those of the storage protein fromcotyledon. A peptide map of the glycoprotein after digestionwith V8 protease was similar to that of the storage protein.Overall, our findings suggest that the glycoprotein recoveredin the nuclear fraction is an isoform of the seed storage proteinthat is expressed only in the mature cells of hypocotyls andepicotyls. ⁴ Present address: Bioscience and Chemistry Division, HokkaidoNational Industrial Research Institute, Agency of IndustrialScience and Technology, Sapporo, 062 Japan     

Promotion of adventitious root formation by 4-chlororesorcinol: A polyphenol oxidase inhibitor

The extent of rooting in cuttings of Phaseolus vulgaris L., and Vigna radiata Wilcz. was affected by 4-chlororesorcinol, a polyphenol oxidase inhibitor. More root primordia and more roots were formed after 4-chlororesorcinol treatment both with and without 10^-5M Indole butyric acid. Promotion of rooting was observed also in cuttings of Elaeagnus pungens, Gypsophilia elegans and Kalanchoe blossfeldiana. The enhancement in bean and mung bean was accompanied by a concomitant wider spatial distribution of the primordia and the resulting adventitious roots. The formation of primordia in the treated cuttings was delayed by 12–24 hours, compared to untreated cuttings. The treatment was effective only when given during the first hours after the preparation of the cutting of bean and mung bean, suggesting involvement in the initiation stage. Hypocotyl extracts of mung bean cuttings, pretreated with 4-chlororesorcinol, exhibited reduced polyphenol oxidase activity. The inhibition was not reversed by washing of the treated extract in 50% acetone or by an overnight dialysis, suggesting tight or maybe even irreversible binding of the inhibitor to the enzyme.Abbreviations 4-CR 4-chlororesorcinol - IBA Indole butyric acid - PPO polyphenol oxidase     

Novel inhibitors of ethylene production in higher plants

Of a number of O-substituted hydroxylamine derivatives, N-benzyloxycarbonyl-L-a-aminooxy-propionicacid and -aminooxyacetic acid inhibited ethylene productionby etiolated mung bean hypocotyls by 50% at 3 and 6 µmconcentrations, respectively. Their potency is thus similarto that of aminoethoxyvinylglycine (50% inhibition at 2 µM),the most potent inhibitor of ethylene production hitherto known.Methionine partially alleviated inhibition of ethylene productionby a-aminooxy-acetic acid. The results are in agreement withthe postulated involvement of pyridoxal phosphate in ethylenebiosynthesis. (Received August 31, 1979; )     

Modulation of the Activity of Purified Tonoplast H+-ATPase from Mung Bean (Vigna radiata L.) Hypocotyls by Various Lipids

H⁺-ATPase was solubilized from the tonoplast of mung bean (Vignaradiata L.) hypocotyls and purified by fast protein liquid chromatographyon a Mono Q ion-exchange column. The purified ATPase hardlycontained any phospholipid, but it did contain 10 to 15 moleculesof sterol and 25 to 30 molecules of glycolipid per ATPase molecule,and it had little activity without exogenously added phospholipids.Each individual polar head group, acylglyceride and fatty acidthat constituted a phospholipid was incapable by itself of activatingthe ATPase. Sterols and cerebroside had little activating effect.Maximal activation of ATPase was noted with asolectin or variousmolecular species of phosphatidylcholine (PC) at 0.005% to 0.01%(w/v). The activation by the various molecular species of PCwas dependent on the length and degree of unsaturation of fattyacyl chains. PC with two saturated and long fatty acyl chainsof more than 18 carbon atoms failed entirely to activate theATPase. PC, PS and PG with 1-palmitoyl (16:0)-2-oleoyl(18:1)fatty acyl chains all activated ATPase to nearly the same extentas asolectin, but the activation by PE and PA with the samefatty acyl composition was 52% and 15% of that by asolectin,respectively. The molecular species of PC with phase-transitiontemperatures below 50C activated ATPase, as determined at 38C.The dependence on temperature of the activation by the molecularspecies of PC indicated that the activation of the ATPase beganclose to the temperature of the phase transition of the PC added.These data indicate that phospholipids in the liquid-crystallinephase are essential for the catalytic activity of the ATPase. (Received June 4, 1992; Accepted January 18, 1993)     

Duroquinone-stimulated NADH oxidase and b type cytochromes in the plasma membrane of cauliflower and mung beans

Microsomal membrane preparations of cauliflower inflorescences and mung bean hypocotyls possess duroquinone (DQ)-stimulated NADH oxidase activities at rates of 1–10 nmol NADH · min⁻ · mg⁻. These redox reaction are associated with the endoplasmic reticulum (ER) and the plasma membrane (PM) as shown by the distributions of marker enzymes in sucrose gradients. The NADH oxidase thus partially cosediments with a specific blue light (or ascorbate) reducible b type cytochrome of the PM.Cauliflower membranes are further purified by means of an aqueous polymer two phase method. The NADH oxidase in this presumptive PM fraction is to some extent stimulated by Triton X-100 and insensitive to KCN (1 mM) or quinacrine (0.4 mM). Kinetics for DQ stimulation showed a biphasic saturation curve. These membranes also have a high FeCN reduction capacity induced by NADH but insensitive to DQ.No evidence could be found in the present study for the involvement of the specific b type cytochrome in the NADH dehydrogenase system.